1. Field of the Invention
The present invention relates to a Trigonopsis variabilis transformed with a gene capable of producing D-amino acid oxidase (hereinafter the DAO gene) and a process for transforming Trigonopsis variabilis. The transfomant of Trigonopsis variabilis is used for producing derivatives of cephalosporin C.
2. Description of Related Art
D-amino acid oxidase (hereinafter DAO) is an enzyme which catalyzes oxidative deaminination of D-amino acids. DAO is also useful to separate L-amino acids from a racemic mixture of DL-amino acids, to produce keto acids from D-amino acids and to analyze D-amino acids. A particular species of DAO produced from the yeast Trigonopsis variabilis (hereinafter T. variabilis) promotes the oxidation of not only D-amino acids but also the antibiotic cephalosporin C to produce 7-.beta.-(5-carboxy-5-oxopentaneamide)cephalosporanic acid, which can be reacted with H.sub.2 O.sub.2 to produce 7-.beta.-(4-carboxybutaneamide)cephalosporanic acid. Cephalosporanic acids are intermediate materials for the synthesis of cephalosporin antibiotics, which are important medical and pharmaceutical products. For example, 7-aminocephalosporanic acid (7ACA), a highly useful intermediate material, is produced by reacting these compounds with H.sub.2 O.sub.2 or a cephalosporin acylase.
Past efforts of others aimed at obtaining increased amounts of DAO investigated various medium and culture conditions: F. M. Huber, BIOTECHNOLOGY LETTERS, 14 (3), 195-200 (1992) and Japanese Patent Publication No. 35118/1980 discloses T. variabilis which is activated by freezing and thawing at pH 3-4, or treating with organic solvents, surfactants and the like. Others have used conventional genetic techniques. For example, international Publication No. W090/12110 discloses a mutant strain of T. variabilis which is obtained by random mutation and produces increased amount of DAO. European Patent Unexamined Publication No. 409521 discloses a problem common to all prior art efforts, namely, that T. variabilis has an esterase which deacetylates 3-positions in cephalosporin C, 7-.beta.-(5-carboxy-5-oxopentaneamide)cephalospranic acid and 7-.beta.-(4-carboxybutaneamide)cephalosporanic acid which esterase reduces the yield by deacetylating the product.
Previously, the present inventors provided a transformant of Escherichia coli (hereinafter E. coli) described in Japanese Patent Application Laid-Open No. 71180/1988, by introducing the DAO gene derived from T. variabilis into a host cell of E. coli whose cephalosporinase activity was lowered. However, the transformed E. coli have to be disrupted before use to recover the DAO from the cells so that their industrial scale employment is impractical. In addition, there remained a residual .beta.-lactamase activity in the transformed E. coli which reduced yields.
Japanese Patent Application Laid-Open No. 200181/1990 and T. Isogai et al., Bio/Technology, 9, 188 (1991) disclose, respectively, transformants of E. coli and Achremonium chrysogenum (hereinafter A. chrysogenum) which were obtained by introducing the DAO gene derived from Fusarium solani. However, the use of these transformants requires taking additional steps to reduce the activity of the endogenous .beta.-lactamase to avoid loss of the end-product.
Thus, heretofore, neither a transformant system using T. variabilis as a host cell nor any transformed cell which does not have endogenous .beta.-lactamase or esterase activity has been developed.